I am using real-time PCR to firstly show the presence/absence of certain targets in my tissue and also to then give more information of the relative amounts of these targets compared to each other. All of my tissue samples are untreated, naive samples so I cannot use a comparative Ct method as I do not have a reference sample. I have thought about using spleen tissue as a reference sample but this won't give me information on levels within my specific tissue.
I could just run a gel, but ideally I would like to compare relative amounts of each target within my tissue.
Hey Martin
So one way you can do this is to use the "deltaCt" method comparing your gene of interest to the housekeeping gene. This way, you can easily arrive at a situation where you can say (for example)… there is negligible expression of gene x, except in the stomach", or most tissues showed 0.001x the GAPDH expression, but there was 0.1x in stomach, 100-fold more"
So, it is easy to compare expression between tissues without having a "treatment" group per se.
You can use a similar method to compare the relative (note, not absolute) expression of different genes in the *same* tissue, just by comparing them all against the same housekeeping gene - this gives you their relative expression to one-another. To do this however, you *must* know that the efficiency of the various reactions all fall within the same parameters - usually 90% - 110%.
You should consider using the method of Pfaffel (sp?) to calculate your DeltaCt, since it takes account of the various reaction efficiencies.
What you *cannot* do is to run a gel and do (say) scanning densitometry, because the curves for fluorescence with (say) EtBr are steep and top out quickly so without having a standard curve (as per ELISA) you have no way whatsoever to know whether your band is saturated or not.
Good Luck!
G
ps is Bill Walker still there? say hello from me!
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